Experimental Study On Effect Of Glaucescent Fissistigma Root Injection On Bel-7402 Cell Proliferation And Angiogenesis

1.Objectives:Recently, the anti-tumor angiogenesis tumor therapy, which is the most promising treatment of tumor targeted therapy, has become a basic and clinical research hot spots. Compared with the traditional treatment of cancer, the most prominent feature of anti-angiogenesis therapy is that it not only is well specific, but also overcomes the deficient of drug resistance and the difficulty of medicine reaching neoplastic tissue .The history of the treatment of the traditional Chinese medicine on angiogenesis is very long in time . With different specificity of Western medicine it shows a great grace. Therefore, research on anti-angiogenesis therapy in the area of the treatment of cancer by traditional Chinese medicine is very significant in the territories of elucidating the basic theory of traditional Chinese medicine, clinical treatment and research on Chinese medicine.Glaucescent Fissistigma Root, also known as Marsdenia tenacissima(Roxb.)Wight et Arn, which is bitter, cool and a little sweet,mainly grows in Yunnan and Guizhou province.According to records,it was first recorded in the "Materia Medica polyanthum" . It can stop coughing and gasping ,eliminate inflammation,relieve pain and has the effect of lactogenesis and diuresis.It was used to treat chronic bronchitis. People obtain fine effect in the process of using Glaucescent Fissistigma Root treating cancers. So it draws attention by many Chinese medicine practitioners. This test is to study and prove that traditional Chinese medicine Glaucescent Fissistigma Root can treat tumor.We investigate the mechanism of anti-tumor from the facts of vascular endothelial growth factor , basic fibroblast growth factor, MMP-2 and p53 .2.Methods:2.1 MTT methodApply MTT deoxidized method to survey the rate of Glaucescent Fissistigma Root inhibiting tumor cell in vitro. Hepatoma carcinoma cell strain Bel-7402 is effected by different density of Glaucescent Fissistigma Root.We get dosage-effectiveness atlas by the OD.2.2 PI single-staining methodApply flow cytometry to detect cell cycle and apoptosis cell population of Bel-7402 cells effected by different concentrations of Glaucescent Fissistigma Root.2.3 ELISA methodApply ELISA Kit to detect the expression of VEGF of Bel-7402 cells in different time effected by different concentrations of Glaucescent Fissistigma Root.2.4 Immunocytochemical methodApply Immunocytochemical method to detect the positive expression rate of b-FGF and MMP-2 protein of Bel-7402 cells effected by different concentrations of Glaucescent Fissistigma Root.2.5 Flow cytometry detect anti-oncogene-p53 proteinumApply flow cytometry to survey p53 proteinum transmutation expressed by Bel-7402 cells, through indirect fluorescent labelling method.P53 proteinum Fluoresence Index(FI) express quantitative p53 proteinum. Green fluoresence LOG collect fluorescence intensity peak amplitude number, which convert linearity scale. It take peak amplitude number of contrast control tube as negative expression FI=1. FI calculated formula as follow: FI= mean fluorecence intensity of detector tube cell / mean fluorecence intensity of detector tube cell contrast control tube. For instance FI>1.0 for masculine expression, FI≤1.0 for negative expression.3.Results:3.1 Four densities of Glaucescent Fissistigma Root(10,20,40mg/ml) have determinate depressant effect on Bel-7402 cells.And the effection is dose-time-dependent. It is significant through statistics analysising discrepancy(P<0.01).3.2 A marked increase in the apoptosis rate of human liver cancer cells Bel-7402 by different concentrations of Glaucescent Fissistigma Root(20,40,80mg/ml) appeared. It is significant through statistics analysising discrepancy.However, there is little change in the cell cycle, which did not confirm that cells were arrested at a specific cycle.3.3 Apply ELISA Kit to detect the expression of VEGF in human liver cancer cell Bel-7402 effected by different concentrations of Glaucescent Fissistigma Root(10,20,40mg/ml). With the increase in drug concentration, VEGF expression gradually reduced.Compared with the negative control group,it is significant.3.4 The result of immunocytochemistry showed that compared with the negative control group,the expression of bFGF and MMP-2 of Bel-7402 cells effected by different concentrations of Glaucescent Fissistigma Root(10,20,40mg/ml) has been reduced.3.5 Utilizing flow cytometry immunofluorescence technic detect p53 gene product, along with the accrescence of medicine density, p53 peak offset rightward overall. According to FI formula calculation, it could calculate FI numerical value. The tumor cells are effected by different concentrations of Glaucescent Fissistigma Root(10,20,40mg/ml) for 24 hours, p53 proteinum in them all show masccline expression,and the FI numerical value increase along with the accrescence of dosage.4.Conclusion: Glaucescent Fissistigma Root has a strong anti-tumor effects in vitro.It can inhibit VEGF, b-FGF, MMP-2 and other substances in the expression of angiogenesis. The mechanism was probably related to the activation of tumor suppressor gene p53 and inhibition of other angiogenic factors, inhibiting tumor cell and endothelial cell proliferation and inducing apoptosis of tumor cells to block tumor angiogenesis.